The interest towards ferulic acid decarboxylase (FDC), piqued by the enzyme’s unique 1,3-dipolar cycloaddition mechanism and its atypic prFMN cofactor, provided several applications of the FDC mediated decarboxylations, such as the synthesis of styrenes, or its diverse derivatives, including 1,3-butadiene and the enzymatic activation of C-H bonds through the reverse carboligation reactions. While rational design-based protein engineering was successfully employed for tailoring FDC towards diverse substrates of interest, the lack of high-throughput FDC-activity assay hinders its directed evolution-based protein engineering. Herein we report a toolbox, useful for the directed evolution based and/or structure-guided protein engineering of FDC, which was validated representatively on the well described FDC, originary from Saccharomyces cerevisiae (ScFDC). Accordingly, the developed fluorescent plate-assay allows in premiere the FDC-activity screens of a mutant library in a high-throughput manner. Moreover, using the plate-assay for the activity screens of a rationally designed 23-membered ScFDC variant library against a substrate panel comprising of 16, diversely substituted cinnamic acids, revealed several variants of improved activity. The superior catalytic properties of the hits revealed by the plate-assay, were also supported by the conversion values from their analytical scale biotransformations. The computational results further endorsed the experimental findings, showing inactive binding poses of several non-transformed substrate analogues within the active site of the wild-type ScFDC, but favorable ones within the catalytic site of the variants of improved activity. The results highlight several ‘hot-spot’ residues involved in substrate specificity modulation of FDC, such as I189, I330, F397, I398 or Q192, of which mutations to sterically less demanding residues increased the volume of the active site, thus facilitated proper binding and increased conversions of diverse non-natural substrates. Upon revealing which mutations improve the FDC activity towards specific substrate analogues, we also provide key for the rational substrate-tailoring of FDC.

Phenylalanine ammonia-lyases (PALs) catalyse the non-oxidative deamination of L-phenylalanine to trans-cinnamic acid, while in the presence of high ammonia concentration, the synthetically attractive reverse reaction occurs. Although they have been intensively studied, the wider application of PALs for the large scale synthesis of non-natural amino acids is still rather limited, mainly due to the decreased operational stability of PALs under the high ammonia concentration conditions of ammonia addition. Herein, we describe the development of a highly stable and active immobilized PAL-biocatalyst obtained through site-specific covalent immobilization onto single-walled carbon nanotubes (SWCNTs), employing maleimide/thiol coupling of engineered enzymes containing surficial Cys residues. The immobilization method afforded robust biocatalysts (by strong covalent attachment to the support) and allowed modulation of enzymatic activity (by proper selection of binding site, controlling the orientation of the enzyme attached to the support). The novel biocatalysts were investigated in PAL-catalyzed reactions, focusing on the synthetically challenging ammonia addition reaction. The optimization of the immobilization (enzyme load) and reaction conditions (substrate : biocatalyst ratio, ammonia source, reaction temperature) involving the best performing biocatalyst SWCNTNH2-SS-PcPAL was performed. The biocatalyst, under the optimal reaction conditions, showed high catalytic efficiency, providing excellent conversion (c ∼90% in 10 h) of cinnamic acid into L-Phe, and more importantly, possesses high operational stability, maintaining its high efficiency over >7 reaction cycles. Moreover, the site-specifically immobilized PcPAL L134A/S614C and PcPAL I460V/S614C variants were successfully applied in the synthesis of several L-phenylalanine analogues of high synthetic value, providing perspectives for the efficient replacement of classical synthetic methods for L-phenylalanines with a mild, selective and eco-friendly enzymatic alternative.

Phenylalanine ammonia-lyases (PALs) catalyse the non-oxidative deamination of l-phenylalanine to trans-cinnamic acid, while in the presence of high ammonia concentration the reverse reaction occurs. PALs have been intensively studied, however, their industrial applications for amino acids synthesis remained limited, mainly due to their decreased operational stability or limited substrate specificity. The application of extensive directed evolution procedures to improve their stability, activity or selectivity, is hindered by the lack of reliable activity assays allowing facile screening of PAL-activity within large-sized mutant libraries. Herein, we describe the development of an enzyme-coupled fluorescent assay applicable for PAL-activity screens at whole cell level, involving decarboxylation of trans-cinnamic acid (the product of the PAL reaction) by ferulic acid decarboxylase (FDC1) and a photochemical reaction of the produced styrene with a diaryltetrazole, that generates a detectable, fluorescent pyrazoline product. The general applicability of the fluorescent assay for PALs of different origin, as well as its versatility for the detection of tyrosine ammonia-lyase (TAL) activity have been also demonstrated. Accordingly, the developed procedure provides a facile tool for the efficient activity screens of large mutant libraries of PALs in presence of non-natural substrates of interest, being essential for the substrate-specificity modifications/tailoring of PALs through directed evolution-based protein engineering.

Phenylalanine ammonia-lyases (PALs) are attractive biocatalysts for the stereoselective synthesis of non-natural phenylalanines. The rational design of PALs with extended substrate scope, highlighted the substrate specificity-modulator role of residue I460 of Petroselinum crispum PAL. Herein, saturation mutagenesis at key residue I460 was performed in order to identify PcPAL variants of enhanced activity or to validate the superior catalytic properties of the rationally explored I460V PcPAL compared with the other possible mutant variants. After optimizations, the saturation mutagenesis employing the NNK-degeneracy generated a high-quality transformant library. For high-throughput enzyme-activity screens of the mutant library, a PAL-activity assay was developed, allowing the identification of hits showing activity in the reaction of non-natural substrate, p-MeO-phenylalanine. Among the hits, besides the known I460V PcPAL, several mutants were identified, and their increased catalytic efficiency was confirmed by biotransformations using whole-cells or purified PAL-biocatalysts. Variants I460T and I460S were superior to I460V-PcPAL in terms of catalytic efficiency within the reaction of p-MeO-Phe. Moreover, I460T PcPAL maintained the high specificity constant of the wild-type enzyme for the natural substrate, l-Phe. Molecular docking supported the favorable substrate orientation of p-MeO-cinnamic acid within the active site of I460T variant, similarly as shown earlier for I460V PcPAL (PDB ID: 6RGS).

A novel phenylalanine ammonia-lyase of the psychrophilic yeast Pseudozyma antarctica (PzaPAL) was identified by screening microbial genomes against known PAL sequences. PzaPAL has a significantly different substrate binding pocket with an extended loop (26 aa long) connected to the aromatic ring binding region of the active site as compared to the known PALs from eukaryotes. The general properties of recombinant PzaPAL expressed in E. coli were characterized including kinetic features of this novel PAL with l-phenylalanine (S)-1a and further racemic substituted phenylalanines rac-1b-g,k. In most cases, PzaPAL revealed significantly higher turnover numbers than the PAL from Petroselinum crispum (PcPAL). Finally, the biocatalytic performance of PzaPAL and PcPAL was compared in the kinetic resolutions of racemic phenylalanine derivatives (rac-1a-s) by enzymatic ammonia elimination and also in the enantiotope selective ammonia addition reactions to cinnamic acid derivatives (2a-s). The enantiotope selectivity of PzaPAL with o-, m-, p-fluoro-, o-, p-chloro- and o-, m-bromo-substituted cinnamic acids proved to be higher than that of PcPAL.

The biocatalytic synthesis of l- and d-phenylalanine analogues of high synthetic value have been developed using as biocatalysts mutant variants of phenylalanine ammonia lyase from Petroselinum crispum (PcPAL), specifically tailored towards mono-substituted phenylalanine and cinnamic acid substrates. The catalytic performance of the engineered PcPAL variants was optimized within the ammonia elimination and ammonia addition reactions, focusing on the effect of substrate concentration, biocatalyst:substrate ratio, reaction buffer and reaction time, on the conversion and enantiomeric excess values. The optimal conditions provided an efficient preparative scale biocatalytic procedure of valuable phenylalanines, such as (S)-m-methoxyphenylalanine (Y=40%, ee>99%), (S)-p-bromophenylalanine (Y=82%, ee>99%), (S)-m-(trifluoromethyl)phenylalanine (Y=26%, ee>99%), (R)-p-methylphenylalanine, (Y=49%, ee=95%) and (R)-m-(trifluoromethyl)phenylalanine (Y=34%, ee=93%).

Modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) enables increased activity and selectivity towards phenylalanines and cinnamic acids mono-substituted with both electron donating (-CH3, -OCH3) and electron withdrawing (-CF3, -Br) groups at all positions (o-, m-, p-) of their aromatic ring. The results reveal specific residues involved in accommodating substituents at o-, m-, p-positions of the substrate’s phenyl ring. The predicted interactions were validated by crystallographic analysis of the binding mode of para-methoxy cinnamic acid complexed at the active site of PcPAL. The biocatalytic utility of the tailored PcPAL mutants was demonstrated by the efficient preparative scale synthesis of (S)-m-bromo-phenylalanine (ee: > 99%, yield: 60%) and (R)-p-methyl-phenylalanine (ee: 97%, yield: 49%), using the corresponding ammonia addition and ammonia elimination reactions catalyzed by the L134A and I460V PcPAL variants, respectively. Overall, the results reveal the potential for structure based protein engineering of PALs to provide enzymes with enhanced catalytic properties and which are specifically tailored for differently substituted phenylalanine analogues of high synthetic value.

Ferulic acid decarboxylase from Saccharomyces cerevisiae (ScFDC1) was described to possess a novel, prenylated flavin mononucleotide cofactor (prFMN) providing the first enzymatic 1,3-dipolar cycloaddition mechanism. The high tolerance of the enzyme towards several non-natural substrates, combined with its high quality, atomic resolution structure nominates FDC1 an ideal candidate as flexible biocatalyst for decarboxylation reactions leading to synthetically valuable styrenes. Herein the substrate scope of ScFDC1 is explored on substituted cinnamic acids bearing different functional groups (–OCH3, –CF3 or –Br) at all positions of the phenyl ring (o−, m−, p−), as well as on several biaryl and heteroaryl cinnamic acid analogues or derivatives with extended alkyl chain. It was found that E. coli whole cells expressing recombinant ScFDC1 could transform a large variety of substrates with high conversion, including several bulky aryl and heteroaryl cinnamic acid analogues, that characterize ScFDC1 as versatile and highly efficient biocatalyst. Computational studies revealed energetically favoured inactive binding positions and limited active site accessibility for bulky and non-linear substrates, such as 2-phenylthiazol-4-yl-, phenothiazine-2-yl- and 5-(4-bromophenyl)furan-2-yl) acrylic acids. In accordance with the computational predictions, site-directed mutagenesis of residue I330 provided variants with catalytic activity towards phenothiazine-2-yl acrylic acid and provides a basis for altering the substrate specificity of ScFDC1 by structure based rational design.

Tailored mutants of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) were created and tested in ammonia elimination from various sterically demanding, non-natural analogues of phenylalanine and in ammonia addition reactions into the corresponding (E)-arylacrylates. The wild-type PcPAL was inert or exhibited quite poor conversions in both reactions with all members of the substrate panel. Appropriate single mutations of residue F137 and the highly conserved residue I460 resulted in PcPAL variants that were active in ammonia elimination but still had a poor activity in ammonia addition onto bulky substrates. However, combined mutations that involve I460 besides the well-studied F137 led to mutants that exhibited activity in ammonia addition as well. The synergistic multiple mutations resulted in substantial substrate scope extension of PcPAL and opened up new biocatalytic routes for the synthesis of both enantiomers of valuable phenylalanine analogues, such as (4-methoxyphenyl)-, (napthalen-2-yl)-, ([1,1'-biphenyl]-4-yl)-, (4'-fluoro-[1,1'-biphenyl]-4-yl)-, and (5-phenylthiophene-2-yl)alanines.

A number of class I lyase-like enzymes, including aromatic ammonia-lyases and aromatic 2,3-aminomutases, contain the electrophilic 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) catalytic moiety. This study reveals that Pseudomonas fluorescens R124 strain isolated from a nutrient-limited cave encodes a histidine ammonia-lyase, a tyrosine/phenylalanine/histidine ammonia-lyase (XAL), and a phenylalanine 2,3-aminomutase (PAM), and demonstrates that an organism under nitrogen-limited conditions can develop novel nitrogen fixation and transformation pathways to enrich the possibility of nitrogen metabolism by gaining a PAM through horizontal gene transfer. The novel MIO enzymes are potential biocatalysts in the synthesis of enantiopure unnatural amino acids. The broad substrate acceptance and high thermal stability of PfXAL indicate that this enzyme is highly suitable for biocatalysis.

Aromatic amino acid ammonia-lyases and aromatic amino acid 2,3-aminomutases contain the post-translationally formed prosthetic 3,5-dihydro-4-methylidene-5H-imidazol-5-one (MIO) group. MIO enzymes catalyze the stereoselective synthesis of α- or β-amino acid enantiomers, making these chemical processes environmentally friendly and affordable. Characterization of novel inhibitors enables structural understanding of enzyme mechanism and recognizes promising herbicide candidates as well. The present study found that both enantiomers of the aminophosphonic acid analogue of the natural substrate phenylalanine and a novel derivative bearing a methylidene at the β-position inhibited phenylalanine ammonia-lyases (PAL), representing MIO enzymes. X-ray methods unambiguously determined the absolute configuration of all tested enantiomers during their synthesis. Enzyme kinetic measurements revealed the enantiomer of the methylidene-substituted substrate analogue as being a mirror image relation to the natural l-phenylalanine as the strongest inhibitor. Isothermal titration calorimetry (ITC) confirmed the binding constants and provided a detailed analysis of the thermodynamic driving forces of ligand binding. Molecular docking suggested that binding of the (R)- and (S)-enantiomers is possible by a mirror image packing.

This study describes the cloning of the gene encoding a novel phenylalanine ammonia-lyase from Kangiella koreensis (KkPAL) into pET19b expression vector. Optimization of protein expression and purification conditions yielded 15 mg pure soluble protein from one liter of E. coli culture. Enzymatic activity measurements of the ammonia elimination reaction from different natural aromatic amino acids proved the protein to be a phenylalanine ammonia-lyase. The isolated protein showed remarkably high, 81.7 °C melting temperature, making it especially suitable for biocatalytic applications.